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Competitive Binding Assay Protocol

The competitive binding assay is an experiment that determines the binding capacity of proteins or chemicals for specialized proteins found on a cell surface, called receptors. The method is often used in medical research but is also able to determine which drugs bind to receptors and modify receptor activity, which in turn can alter cell behavior.

  1. Cell Preparation

    • Re-suspend cells into ice-cold phosphate buffered saline (PBS). Measure protein concentration to confirm equal protein levels by using a specialized commercially available protein assay.

    Assay

    • Add a radioactivity-labeled protein known to bind to the receptor, the ligand, to the cell or tissue suspension. Add the competitor, which is the test drug or protein under study and incubate with the radioactive ligand and cell suspension. A range of concentrations of the test drug should be studied. Following incubation, separate bound and unbound ligand and measure radioactivity.

    Data Analysis

    • The usual measure to be determined is half the maximum concentration that inhibits the receptor otherwise known as IC50. This is calculated by plotting the concentrations on a logarithmic scale on the X axis of a graph while the Y axis represents the percentage of inhibition; a sigmoid curve, which is an S-shape, should be generated. The IC50 is the 50 percent inhibition point, and the corresponding concentration can be read from the graph.

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